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2575 Detection of Novel Pathogenic Gene Rearrangements in Pediatric Acute Myeloid Leukemia By RNA Sequencing

Acute Myeloid Leukemia: Biology, Cytogenetics and Molecular Markers in Diagnosis and Prognosis
Program: Oral and Poster Abstracts
Session: 617. Acute Myeloid Leukemia: Biology, Cytogenetics and Molecular Markers in Diagnosis and Prognosis: Poster II
Sunday, December 6, 2015, 6:00 PM-8:00 PM
Hall A, Level 2 (Orange County Convention Center)

Norio Shiba, MD, PhD1*, Kenichi Yoshida, MD, Ph.D.2*, Yuichi Shiraishi, MD, Ph.D.3*, Yusuke Hara, MD1*, Genki Yamato, MD1*, Taeko Kaburaki4*, Masashi Sanada, MD, Ph.D.5*, Kentaro Oki, MD, PhD6*, Daisuke Tomizawa, MD, PhD7, Manabu Sotomatsu8*, Hirokazu Arakawa9*, Keizo Horibe, MD, PhD10, Myoung-Ja PARK11*, Takashi Taga, MD, PhD12*, Souichi Adachi, MD, PhD13, Akio Tawa, MD, PhD14*, Satoru Miyano, MD, Ph.D.3*, Seishi Ogawa, MD, Ph.D.2 and Yasuhide Hayashi, MD, PhD15

1Department of Pediatrics, Gunma University Graduate School of Medicine, Maebashi, Japan
2Department of Pathology and Tumor Biology, Graduate School of Medicine, Kyoto University, Kyoto, Japan
3Human Genome Center, Institute of Medical Science, The University of Tokyo, Tokyo, Japan
4Gunma Unversity Graduste School of Medicine, Maebashi, Japan
5Department of Advanced Diagnosis, Clinical Research Center, National Hospital Organization Nagoya Medical Center, Nagoya, Japan
6Gunma Children's Medical Center, Shibukawa, Japan
7Division of Leukemia and Lymphoma, Children’s Cancer Center, National Center for Child Health and Development, Tokyo, Japan
8Hematology/Oncology, Gunma Children's Medical Center, shibukawa, Japan
9Pediatrics, Gunma Unversity Graduste School of Medicine, Maebashi, Japan
10Clinical Research Center, National Hospital Organization Nagoya Medical Center, Nagoya, Japan
11Hematology/Oncology, Gunma Children's Medical Center, Shibukawa, Japan
12Department of Pediatrics, Shiga University of Medical Science, Otsu, Japan
13Department of Human Health Sciences, Kyoto University, Kyoto, Japan
14Department of Pediatrics, National Hospital Organization, Osaka National Hospital, Osaka, Japan
15Department of Hematology/Oncology, Gunma Children's Medical Center, Shibukawa, Japan

Background

Pediatric acute myeloid leukemia (AML) comprises approximately 20% of pediatric leukemia cases. AML is a major therapeutic challenge in pediatric oncology, and the current overall survival rate is <70%. The pathogenesis of AML is heterogeneous, and causes include various chromosomal aberrations, gene mutations/epigenetic modifications, and deregulated/overregulated gene expression, resulting in increased proliferation and decreased hematopoietic progenitor cell differentiation. Recurrent chromosomal structural aberrations such as t(8;21), inv(16), and MLL-rearrangements are well established as diagnostic and prognostic markers in AML. Furthermore, recurrent mutations in FLT3, KIT, and RAS have been reported in both adult and pediatric AML. Recently, massively parallel sequencing has facilitated the discovery of recurrent mutations in DNMT3A, TET2, and IDH, which are clinically useful for predicting the prognosis. However, these mutations are rare in pediatric AML, thereby suggesting that other genetic alterations may exist in pediatric AML.

 In addition, recent studies have reported that the NUP98–NSD1 fusion is an adverse AML prognostic marker and that PRDM16 (also termed MEL1) is a representative gene that is overexpressed in patients who have the NUP98–NSD1 fusion. PRDM16 overexpression occurs in nearly a quarter of pediatric AML patients who are NUP98–NSD1 negative, and this overexpression is increased in specimens with other high-risk lesions (e.g., FLT3–ITD, NUP98–NSD1, and MLL–PTD).

Patients and Methods

To obtain a complete overview of gene rearrangements and other genetic lesions, we performed RNA sequencing of samples from 47 de novo pediatric AML patients using Illumina HiSeq 2000, including 39 patients with normal karyotypes and 6 patients with Trisomy 8. Among these 47 patients, 35 patients overexpressed PRDM16, which was strongly associated with a poor prognosis in our previous studies. As a control, we selected 12 patients with low PRDM16expression levels. All patients were enrolled and treated as part of the AML-05 study conducted by the Japan Pediatric Leukemia/Lymphoma Study Group. We determined the known gene mutations present in these patients using the RNA sequencing data.

Results

Approximately 300 candidate gene rearrangements were identified in 46/47 samples, including 26 in-frame and 78 out-of-frame gene rearrangements. Several recurrent gene rearrangements identified in this study involved previously reported targets in AML, such as FUS–ERG, NUP98–NSD1, and MLL–MLLT3. However, several novel gene rearrangements were identified in the current study, including HOXA10–HOXA-AS3, PRDM16–XXX, CUL1–YYY, and DAZAP1–ZZZ. At present, we are validating these novel gene rearrangements using Sanger sequencing. Known gene alterations, such as FLT3–ITD, MLL–PTD, and mutations of RAS, KIT, CEBPA, WT1, and NPM1genes, were detected by RNA sequencing.

Conclusion

In the present study, RNA sequencing was employed to elucidate the complexity of gene rearrangements/mutations in pediatric AML genomes. Our results indicate that a subset of pediatric AML represents a discrete entity that can be discriminated from adult AML in terms of the spectrum of gene rearrangements/mutations. We identified at least one potential gene rearrangement or driver mutation in nearly all AML samples, including various novel fusion genes. Thus, our results suggest that gene rearrangements and mutations play essential roles in pediatric AML.

Disclosures: No relevant conflicts of interest to declare.

*signifies non-member of ASH